Method for creating polyploidy loach by knocking out espl1 gene

Abstract

The invention discloses a method for creating loach polyploidy, which comprises the following steps: knocking out a diploid loach espl1 gene by using a CRISPR / Cas9 gene knockout technology, and obtaining espl1 heterozygous mutation diploid loach (espl1 + / -) through genetic typing and breeding; by utilizing the characteristic that the espl1 + / -male can generate unreduced diploid sperms, combining the espl1 + / -male with diploid wild female haploid ovum, so as to obtain about 10% of espl1 heterozygous mutation triploid loach (espl1 + / + / -); the espl1 + / + / -male loach (espl1 + / + / + / -) is combined with a diploid wild female haploid ovum according to the characteristic that the espl1 + / + / -male loach can generate unreduced triploid sperms, and then the espl1 heterozygous mutation tetraploid loach (espl1 + / + / + / -) is obtained; the characteristic that espl1 + / + / + / -can generate normal diploid ovum is utilized to be combined with diploid wild type male haploid sperms, and then a large number of triploid offspring are prepared.

Description

Technical Field

[0001] The invention discloses a method for creating polyploid loach, and belongs to the field of aquatic genetic breeding. Background Art

[0002] Polyploid organisms, due to their significant growth advantages, high population productivity, and excellent disease and stress resistance, far surpass diploids in economic benefits and application potential, demonstrating enormous development value. Furthermore, the sterility of triploids effectively inhibits overproduction in farmed fish, preventing the mixing of germplasm resources and having important implications for ecological conservation. Therefore, the research and application of polyploid fish not only promotes the sustainable development of aquaculture but also becomes a key research area in this field.

[0003] Currently, common methods for inducing polyploidy include hybridization, temperature shock, hydrostatic shock, and chemical treatment. Hybridization involves hybridizing tetraploids with diploids to produce triploids. Temperature shock and hydrostatic shock methods produce triploids or tetraploids by inhibiting the extrusion of the second polar body or the first mitosis. Chemical treatment involves using chemicals such as colchicine to inhibit the extrusion of the second polar body or the first mitosis, resulting in triploids or tetraploids. Despite the multitude of methods currently available for inducing polyploidy, technical bottlenecks such as low triploid induction rates, embryo hatching rates, and juvenile survival rates, as well as the difficulty in obtaining adult fish after tetraploid induction, have severely hampered the large-scale production and application of artificial polyploidy in fish.

[0004] Existing research data show that the development of artificial polyploidy in fish is related to chromosome segregation, and the Extra spindle pole bodies like 1 gene ( spl1 ) encodes separase, an enzyme that primarily catalyzes the dissociation of cohesin during late mitosis, leading to the separation of sister chromatids. By knocking out this gene, artificial mitotic defects can be induced to create polyploidy, providing a new and feasible approach for inducing polyploidy in fish. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for creating polyploid loaches using the espl1 gene, so as to overcome the defects and shortcomings of the above-mentioned background technology and provide solid technical support for the artificial creation of polyploid loaches.

[0006] To achieve the above objectives, the present invention proposes the following technical solutions: A knockout spl1 The method for genetically creating polyploid loach comprises the following steps: 1) Using CRISPR / Cas9 system to modify wild-type diploid loach spl1 The gene was knocked out and diploid loach was obtained through genotyping. spl1 The F0 generation of gene mutants, spl1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; 2) Diploid loach spl1 The F0 generation of gene mutants was hybridized with wild-type diploid loach to obtain the F1 generation. When the offspring were sexually mature, they were screened by genotyping. spl1 Heterozygous diploid loach ( spl1 + / - ); 3) The male spl1 Heterozygous diploid loach ( spl1 + / - ) were hybridized with diploid wild-type female loach to obtain the F2 generation. When the offspring were sexually mature, they were screened by genotyping and ploidy analysis. spl1 Heterozygous triploid loach ( spl1 + / + / - ); 4) Male spl1 Heterozygous triploid loach ( spl1 + / + / - ) were hybridized with diploid wild-type female loach to obtain the F3 generation. When the offspring were sexually mature, they were screened by genotyping and ploidy analysis. spl1 Heterozygous tetraploid loach ( spl1 + / + / + / - ); 5) The female spl1 Heterozygous tetraploid loach ( spl1 + / + / + / - ) were hybridized with diploid wild-type male loaches to obtain a fully triploid population of the F4 generation.

[0007] Among them, the spl1 The gene knockout method includes the following steps: 1) Based on the CRISPR / Cas9 knockout principle, a knockout target site was designed in the loach espl1 gene ORF sequence; 2) In loach spl1 Upstream and downstream primers containing the knockout target site sequence were designed on the gene sequence, and PCR amplification was performed using loach cDNA as a template. After in vitro transcription and purification, gRNA was obtained; 3) Using the linearized Cas9 plasmid as a template, Cas9 mRNA was obtained by in vitro transcription and purification; 4) Microinject gRNA and Cas9 mRNA into diploid loach fertilized eggs, and then incubate and culture the fertilized eggs to obtain diploid loach spl1 Gene mutants.

[0008] Among them, the spl1The gene knockout target site sequence is shown in SEQ ID NO: 2.

[0009] Furthermore, the upstream primer sequence is shown as SEQ ID NO: 3, and the downstream primer sequence is shown as SEQ ID NO: 4.

[0010] The injection concentration of the gRNA is 100 ng / μL, the injection concentration of the Cas9 mRNA is 100 ng / μL, the injection dose is 2.5 nL, and the injection site is the animal pole.

[0011] The present invention also provides a reagent for creating polyploid loach, the reagent comprising Cas9 gene editing protein or its expression vector, and a protein that guides the Cas9 gene editing protein to specifically bind to spl1 The gRNA of the gene or its expression vector, spl1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0012] The beneficial effects of the present invention are: Gene knockout technology to obtain diploids spl1 Genetic mutant loach, using spl1 The genetically modified loach males are able to produce unreduced diploid sperm, which can be used to obtain triploid loaches. spl1 The heterozygous mutant triploid loach can produce unreduced triploid sperm, which can be used to obtain tetraploid loach. spl1 Heterozygous tetraploid loaches are capable of producing large numbers of diploid gametes, allowing for the production of large numbers of triploid loaches. Compared to traditional polyploid induction methods, this method has a certain degree of universality and better genetic controllability, improving the efficiency of artificially inducing polyploid loaches and providing good technical support for the production and application of polyploid loaches. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the process for creating polyploid loach.

[0014] Figure 2 The invention is constructed to obtain spl1 Heterozygous loach mutation type.

[0015] Figure 3 Diploid spl1 + / - ♂ × Ploidy detection peak diagram of the triploid offspring of the diploid WT♀, embryonic chromosome karyotype, and growth comparison diagram of the triploid and normal diploid.

[0016] Figure 4 Triploid spl1+ / + / - Ploidy detection peak diagram of the tetraploid offspring of ♂×diploid WT♀ and growth comparison between the tetraploid and normal diploid.

[0017] Figure 5 Tetraploid spl1 + / + / + / - Ploidy detection peak diagram and embryonic chromosome karyotype of triploid offspring of ♀×diploid WT♂. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the embodiments.

[0019] Example 1 Loach spl1 Gene knockout 1. Design of CRISPR / Cas9 knockout targets Obtained from NCBI database spl1 The genomic nucleic acid sequence of the gene, the basic design principles of the reference target site, in loach spl1 The target site was designed in the third exon region of the gene ( Figure 2 The knockout target site sequence selected in the present invention is GGCCTTGCTAGTCGATTG (as shown in SEQ ID NO: 2). Detection primers were designed around the knockout target site. spl1 -F / R amplifies the fragment containing the target site sequence, where the upstream detection primer spl1 -F sequence is GACTGGTGGAGTTGGCTGA (as shown in SEQ ID NO: 3), downstream detection primer spl1 The -R sequence is ATTCCCCTACCACCTCCT (as shown in SEQ ID NO: 4).

[0020] The enzyme-containing PCR premix of Shanghai Yisheng Biotechnology Co., Ltd. was used for spl1 For PCR amplification of the gene target site fragment, the following reaction system was prepared in a centrifuge tube (total volume 20 μL): 2 xHieff® PCR Master Mix10.0µL PCR-Grade Water 7.0µL Upstream primer esp11 -F1.0µL Downstream primer espI1 -R1.0µL 1.0µL genomic DNA The PCR amplification program was set as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 35 amplification cycles; and final extension at 72°C for 10 min.

[0021] The sequence information of the amplified product is obtained, and then compared with the target site sequence. If the results are the same, it means that the target site is available and the next step can be carried out.

[0022] 2. Preparation of gRNA First, according to the spl1 Prepare the following reaction system in a sterile PCR tube (total volume 50 μL) using the gRNA upstream primer and the matching downstream primer (as shown in SEQ ID NO: 4 and SEQ ID NO: 5) for the gene target site sequence: PCR-Grade Water 32.0µL 10xPCR Buffer 5.0µL dNTP Mixture 5.0µL rTaq 0.5µL Upstream primer gRNA-F (10mmol / L) 2.5µL Downstream primer gRNA-R (10mmol / L) 2.5µL 2.5 µL of cDNA template.

[0023] The PCR amplification reaction conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 30 amplification cycles; and final extension at 72°C for 10 min.

[0024] The amplified gRNA fragments were cleaned and recovered using the AxyPrep PCR Cleanup Kit from AXYGEN. After obtaining the purified gRNA, in vitro transcription of the gRNA was immediately performed using the T7Kit from Ambion. The specific steps are as follows: 1) Prepare the following reaction system in a sterile EP tube (total volume 20.0 μL): 2.5 mmol / L NTP 4.0µL 10x Reaction Buffer 2.0µL Linear Template DNA 5.0µL T7 Enzyme Mix 2.0µL DEPC H2O7.0µL After thorough mixing, incubate in a 37°C water bath for 1 h.

[0025] 2) Add 1.0 μL of TURBO DNase to the tube and treat in a 37°C water bath for 15 minutes to remove excess DNA template. TMThe miRNA Isolation Kit recovers the in vitro transcribed gRNA. The specific steps for recovery and purification are as follows: Dilute gRNA to 300 μL using RNase-free water and add an equal volume of anhydrous ethanol; Transfer the above solution to the recovery column and centrifuge at 10000 r / min for 15s; The filtrate was discarded, 700 μL miRNA Wash Solution I was added, and centrifuged at 10,000 rpm for 10 seconds. Discard the filtrate, add 500 μL Wash Solution II, centrifuge at 10,000 rpm for 10 seconds, and repeat the operation once; The filtrate was discarded and the empty column was centrifuged at 10000 r / min for 1 min to remove the residual liquid in the recovery column; Add an appropriate amount of RNase-free water preheated at 95°C, centrifuge at 13,000 r / min for 30 seconds, collect the gRNA, detect the concentration, and freeze at -80°C for later use.

[0026] 3. Preparation of Cas9 mRNA Linearize the pSP6-2sNLS-spCas9 vector using a single enzyme digestion with XbaI (37°C in a water bath for at least 4 hours). After confirming complete linearization by electrophoresis, directly recover the linearized product. Using the purified linearized Cas9 plasmid as a template, in vitro transcribe Cas9 mRNA, purify it, and store it at -80°C at a concentration of 900 ng / μL.

[0027] 4. In vitro microinjection The night before injection, healthy, well-developed diploid loaches were selected as parents for artificial induction of spawning. The dosage of LRH-A (240 μg / kg) and DOM (4 mg / kg) was used for females, with half the dosage used for males. The parents were then placed in aerated water at 28°C. The following morning, once the females could successfully ovulate, artificial insemination was performed. Fertilized eggs were pipetted onto a microinjection plate. One-cell stage fertilized eggs were microinjected using a microinjector. The final concentrations of Cas9 mRNA and gRNA were 100 ng / μL, with a volume of 2.5 nL per injection. The injection site was the animal pole. After injection, the fertilized eggs were placed in a 28°C constant-temperature, micro-aerated incubator for incubation.

[0028] Example 2 Breeding of polyploid loach 1. Screening of positive individuals in the F0 generation After the fertilized eggs hatched, 5 larvae were randomly selected, and their genomic DNA was extracted for PCR amplification and sent to Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The F0 individuals with double peaks at the target site were considered to be successfully knocked out according to the sequencing results ( Figure 2 ).

[0029] 2. Screening of positive individuals in the F1 generation After the F0 that has been effectively knocked out has grown to sexual maturity, it is mated with the wild type to obtain F1 embryos. The F1 generation is raised to adulthood, the tail fin of the adult fish is cut, and the genomic DNA is extracted as a PCR template. The detection primers are used to spl1 -F / R were PCR amplified, and the amplified products were sent to Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. for Sanger sequencing. spl1 The gene sequence is aligned to exclude the wild type, and the remaining individuals are F1 heterozygotes. The F1 heterozygotes obtained in the present invention are heterozygous mutant loaches with a 1 bp insertion. Compared with the wild type, a base A is inserted at the knockout target site. 3. spl1 Breeding and screening of heterozygous mutant triploid loaches There are two sexes in the F1 generation spl1 Mutants, heterozygous female diploid loaches ( spl1 + / - ) and wild-type male diploid loach; heterozygous male diploid loach ( spl1 + / - ) were hybridized with wild-type female diploid loaches; heterozygous diploid loaches ( spl1 + / - ) Self-pollination to obtain reproductive offspring.

[0030] For analysis spl1 The ploidy composition of the mutant offspring was analyzed using a ploidy analyzer for the offspring of the three reproductive types. The specific process is as follows: right spl1 The DNA content of the knockout mutant progeny is determined as follows: 1) Use a clean pipette to transfer the larvae to sterile EP tubes, one per tube. Add 500 μL of 1× PBS buffer (NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 4.3 mM, KH₂PO₄ 1.4 mM) to the sample tube. Place the pestle into the EP tube containing the sample and rotate it left and right to thoroughly grind. Place the resulting cell suspension on ice until ready to use. 2) Ploidy determination using the relative cellular DNA content method. Before ploidy determination, ploidy calibration was performed using wild-type larvae that had reached the same developmental stage. Before loading the sample, 100 μL of DAPI dye was added to the ground cell suspension, the solution was mixed, and staining was performed for 5 minutes in the dark. The sample was loaded onto the ploidy analyzer for analysis. The peak value of the sample to be tested was compared with the peak value of the wild-type sample as a control to determine the ploidy of the larvae to be tested.

[0031] Perform chromosome karyotype analysis on offspring embryos. The specific steps are as follows: 1) When the embryos begin to accumulate eye pigment, select 10-15 embryos as a replicate, and prepare three replicate treatment groups for statistical analysis. Before processing, carefully remove the zygote with a syringe to avoid damaging the embryo. 2) Using a clean pipette, transfer the embryos to a Petri dish containing 0.1% colchicine solution and soak at room temperature for 45 minutes. 3) Use a syringe to remove the colchicine solution from the culture dish and add 0.8% sodium citrate solution to submerge the embryos. Incubate in a hypotonic solution for 20 minutes, changing the hypotonic solution once during this period. 4) After hypotonicity, fix the embryos in pre-chilled Carnoy's fixative (methanol: glacial acetic acid = 3:1) for 15 minutes each, for a total of three fixation cycles. 5) After fixation, transfer the embryos to a 2.0 mL EP tube filled with fresh fixative and store at -20°C overnight. 6) The next day, discard the fixative solution in the tube and add 1-2 drops of 50% glacial acetic acid to dissociate the sample. Use a syringe to tear the embryo into a single-cell suspension. Add an appropriate amount of freshly prepared Carnoy's fixative and prepare chromosome sections by drop-mounting. Fix the slides with flame and allow to air dry at room temperature. 7) Wright-Giemsa staining, observation and photography under a microscope, and counting of the chromosome numbers of the embryos.

[0032] According to statistics, spl1 + / - In the cross between female × wild-type male, only diploid offspring are produced, which are similar to wild type; spl1 + / - Male × wild-type female hybrid offspring and spl1 + / - 9.82% of the triploids were detected in the self-pollinated offspring. The triploid offspring had 75 chromosomes and a DNA content that was 1.5 times that of the wild type ( Figure 3 ). 4. spl1 Breeding and screening of heterozygous mutant tetraploid loach 3N spl1 + / + / -The male and wild-type female were used as breeding parents and hybridized to obtain offspring embryos. A large number of offspring embryos of this breeding combination died during development, and only a small number of individuals were able to develop normally and survive. The ploidy of the offspring larvae of this breeding combination was tested using a ploidy analyzer, and viable tetraploid offspring were found ( spl1 + / + / + / - )( Figure 4 ), however, such triploid males produce aneuploid sperm, which may induce hyperdiploid and hypertriploid offspring with low viability.

[0033] 5. Mass cultivation of triploid loaches by spl1 + / + / + / - Females were crossed with wild-type males as breeding parents, and offspring embryos were obtained. The offspring embryos developed normally, with a fertilization rate and survival rate exceeding 80%. Ploidy analysis and chromosome counts showed that spl1 + / + / + / - The offspring of female loaches mating with diploid wild-type male loaches are all triploid ( Figure 5 ), which shows that spl1 + / + / + / - Female loaches produce diploid eggs.

[0034] Figure 1 Shows the use of spl1 Pathways to obtain fully triploid populations of mutants.

[0035] Description of Sequence Listing: SEQ ID NO: 1: spl1 Gene sequence; SEQ ID NO: 2: spl1 gene knockout target site sequence; SEQ ID NO: 3, 4: Upstream and downstream primers of target site sequence spl1 -F / R sequence.

Claims

1. A knockout espl1 A method for genetically creating polyploid loach, characterized in that The following steps are involved: 1) Using CRISPR / Cas9 system to modify wild-type diploid loach espl1 The gene was knocked out and diploid loach was obtained through genotyping. espl1 The F0 generation of gene mutants, espl1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; 2) Diploid loach espl1 The F0 generation of gene mutants was hybridized with wild-type diploid loach to obtain the F1 generation. When the offspring were sexually mature, they were screened by genotyping. espl1 Heterozygous diploid loach ( espl1 + / - ); 3) The male espl1 Heterozygous diploid loach ( espl1 + / - ) were hybridized with diploid wild-type female loach to obtain the F2 generation. When the offspring were sexually mature, they were screened by genotyping and ploidy analysis. espl1 Heterozygous triploid loach ( espl1 + / + / - ); 4) Male espl1 Heterozygous triploid loach ( espl1 + / + / - ) were hybridized with diploid wild-type female loach to obtain the F3 generation. When the offspring were sexually mature, they were screened by genotyping and ploidy analysis. espl1 Heterozygous tetraploid loach ( espl1 + / + / + / - ); 5) The female espl1 Heterozygous tetraploid loach ( espl1 + / + / + / - ) were hybridized with diploid wild-type male loaches to obtain a fully triploid population of the F4 generation.

2. The method for producing polyploid loach according to claim 1, wherein: described espl1 The gene knockout method includes the following steps: 1) According to the CRISPR / Cas9 knockout principle, in loach espl1 Design knockout target sites on the gene ORF sequence; 2) In loach espl1 Upstream and downstream primers containing the knockout target site sequence were designed on the gene sequence, and PCR amplification was performed using loach cDNA as a template. After in vitro transcription and purification, gRNA was obtained; 3) Using the linearized Cas9 plasmid as a template, Cas9 mRNA was obtained by in vitro transcription and purification; 4) Microinject gRNA and Cas9 mRNA into diploid loach fertilized eggs, and then incubate and culture the fertilized eggs to obtain diploid loach espl1 Gene mutants.

3. The method for producing polyploid loach according to claim 2, wherein: described espl1 The gene knockout target site sequence is shown in SEQ ID NO:

2.

4. The method for producing polyploid loach according to claim 2, wherein: The upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO:

4.

5. The method for producing polyploid loach according to claim 2, wherein: The injection concentration of the gRNA was 100 ng / μL, the injection concentration of the Cas9 mRNA was 100 ng / μL, the injection dose was 2.5 nL, and the injection site was the animal pole.

6. A reagent for producing polyploid loach, characterized in that: The reagent includes a Cas9 gene editing protein or an expression vector thereof, and a vector that guides the Cas9 gene editing protein to specifically bind to espl1 The gRNA of the gene or its expression vector, espl1 The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

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